Function: Z Lock

Function: Z Lock

The Nanoimager has a Z Lock functionality which allows for long and stable acquisitions from the desired focal plane. This feature is very useful for multiple types of imaging including dSTORM, tracking, overview scan, and multi-position acquisitions. The Z lock function utilizes the focus laser pattern which is projected onto a small camera window below Focus Camera View. The focus module uses a near-infrared laser (850 nm) to avoid bleaching of the sample. The focus laser pattern is unique to each Nanoimager, and an example is shown below:

Instructions

  1. Connect to the microscope and place a bead slide on the microscope with fresh oil.
  2. Lift up the stage to around 200 nm and then gradually lower down the stage until reaching the first focus of the sample (i.e. the inner side of the coverslip).
  3. Click on Set Focus Ref. to set the focus reference/standard. The stage will then be moved up and down to gather the pattern from the focus camera for evaluation.
  4. Once the process is finished, click on OK in the pop-up window and the Focus and Z Lock will appear checkable. In older versions (1.6 or below), a set of curves will be displayed upon acceptance.

  5. Click on Focus to perform a one-time focusing or click on Z Lock to enable real-time focusing.
  6. Set the Z Offset value to "micro-tune" the focus performance after the Z lock function is enabled.

Discussions

  1. The Z lock function is different from "autofocus" since the initial focus measure needs to be done manually.
  2. The Z lock function maintains the focus rather than the position, so often time the Z position is changing while moving the stage along X and/or Y.
  3. High precision #1.5 or #1.5H coverslips are required and the coverslip surface must be clean and scratch-free.
  4. The sample needs to be mounted in the correct imaging medium. The focus pattern may not be visible if there is a refractive index mismatch between the sample and the mounting medium.
  5. Any air bubble in the oil can strongly impact the Z lock performance. Do replenish oil if moving samples over large areas like moving from one to another well of an ibidi chamber as the long motion often spread the oil and reduce the amount of oil between the sample and objective.
If at any point you are having issues with the Z lock function, please do not hesitate to contact the CX team.